Fig 1: The characteristics of a paediatric patient with a DNAH5 mutation. (A) The pedigree structure of the family with DNAH5 mutation. The black squares represent the proband. (B) Sanger sequencing identifying the compound mutations in the patient and mutation-carrier parents. (C) Ultrastructure of the ciliary axonemes from a healthy child and the patient by TEM showing the “9 + 2” structure. The red arrow reflects the partial defect of the outer dynein arm. (D) The results of spirometry in the patient with PCD demonstrating moderate obstructive ventilation dysfunction and small airway airflow obstruction. Pred: predicted value; Act: actual measured value; Act/Pred: ratio of actual measured to predicted values; Sal: measured value after inhaling salbutamol; Improvement: ratio of Sal/Pred to Act/Pred; FVC: forced vital capacity; FEV1: forced expiratory volume in one second; FEV1/FVC: ratio of FEV1 to FVC; FEV1/VC MAX: ratio of FEV1 to VC MAX; PEF: peak expiratory flow rate; MEF 75: maximal expiratory flow after 75% of the FVC has not been exhaled; MEF 50: maximal expiratory flow after 50% of the FVC has not been exhaled; MEF 25: maximal expiratory flow after 25% of the FVC has not been exhaled; MMEF 75/25: maximal mid-expiratory flow. The measurements of FEV1/FVC and FEV1/VC MAX were 84.6% and 84.5%, respectively, suggesting a moderate obstructive ventilation dysfunction. The MEFs of 50 and 25 were less than 65%, suggesting a small airway airflow obstruction. The reference range is shown in brackets; the red boxes indicate abnormal values. (E) The nasopharyngeal CT scan of the nasal sinuses shows pansinusitis in the patient. The red arrow reflects nasosinusitis.
Fig 2: Decreased immune response in DNAH5-mutated organoids. (A) GO enrichment analysis of biological processes in DNAH5-mutated and normal airway organoids. The immune responses were downregulated in DNAH5-mutated airway organoids (left), whereas epithelial cell differentiation was downregulated (right). (B) The heatmap illustrates the expression level changes of the statistically significant proteins related to the adaptive immune response, innate immune response and epithelial development between DNAH5-mutated and normal airway organoids. (C) Stepwise protocol for airway organoids infected by RSV. (D) The copies of RSV-N detected by ddPCR confirming the different infections between DNAH5-mutated and normal airway organoids. * p < 0.05; n = 3 replicates. (E) The expression of immune factors in DNAH5-mutated and normal airway organoids infected by RSV at 48 hpi. **** p < 0.0001; n = 3 replicates.
Fig 3: Patient-specific child airway organoids from a paediatric patient with a DNAH5 mutation. (A) Schematic workflow of child airway organoid generation derived from bronchoscopic biopsy. (B) Bright-field images depicting child airway organoid phenotypes. Scale bar = 100 µm. (C) Whole-mount child airway organoid immunofluorescence shows DNAH5 expression on the cilia. Hoechst 33342, blue; DNAH5, red; acetylated tubulin, green. Scale bar = 10 µm. (D) Western blot analysis showing the protein expression of DNAH5 in airway organoids from PCD patients and healthy controls. Krt5 is a marker protein of basal cells; GAPDH is the endogenous control. (E) SPY555-tubulin live imaging showing the difference in cilia motility between normal and PCD airway organoids. Normal cilia are motile (upper), whereas the cilia in PCD airway organoids with DNAH5 mutation are immotile (lower). The right part is a schematic representation. The white arrows reflect the cilia, and the time was measured in seconds(s). Scale bar = 10 µm. (F) Ciliary beat frequency of normal and PCD airway organoids with DNAH5 mutation. The cilia in PCD airway organoids are immotile. **** p < 0.0001. n = 10 per group.
Fig 4: Single-cell transcriptional profiles from child PCD organoids. (A) UMAP plots of expression from scRNA-Seq of child airway organoids (DNAH5-mutated and control). (B) GO enrichment analysis of biological processes in basal cells (DNAH5 vs. CTRL). (C) Heatmap of the differentially expressed genes in immune response, regulation of cell morphogenesis, cell-substrate adhesion and epithelial cell proliferation between DNAH5 mutation and normal organoids. (D) GO enrichment analysis of biological processes in multiciliated cells (DNAH5 vs. CTRL). (E) Heatmap of the differentially expressed genes in the immune response between DNAH5-mutated and normal organoids. (F,G) Dot plot of Wnt, TGF-β/BMP and Notch pathway-regulated genes in basal cells and multiciliated cells in DNAH5-mutated and normal airway organoids. The colour gradient (grey to blue) and dot size indicate the mean marker expression and the percentage of cells expressing the marker for each cluster.
Supplier Page from Abcam for Anti-DNAH5 antibody